Natural killer (NK) cells develop from primitive hematopoetic stem cells into functional anti-tumor effector cells that express receptors recognizing MHC class I ligands. In addition to the killer immunoglobulin-like receptor (KIR) family, NKG2A and LIR-1 also recognize MHC class I ligands. Our data show that although the population of KIR-/NKG2A- NK cells circulating in blood cannot be inhibited by self ligands, they are not autoreactive, but instead are hyporesponsive and tolerant. The mechanism by which these hyporesponsive cells acquire function is dependent first on the acquisition of NK cell receptors and then on subsequent interactions with their MHC class I ligands. The process of NK cell education is also referred to as licensing, calibration or arming/disarming. NK cells that have been educated by exposure to cognate ligand exhibit higher function against ligand-negative targets, while uneducated NK cells remain hyporesponsive. The overarching hypothesis is that the process of human NK cell education is coordinated with NK cell development and is further modulated by activation-inflammatory signals and inhibitory receptor ligation. We propose that although NK cell education can precisely adjust the innate immune response, this mechanism may not be absolute and that it can be reversed by activation-inflammatory signals. Although interactions between KIR and KIR-ligand dominate the clinical literature, a population of KIR- NK cells in blood exhibit potent anti-tumor function, possibly because they have been educated through NKG2A or LIR-1. During the current funding, we established a mechanistic link between IL-15, known to activate NK cells, and the upregulation of c-Myc. C-Myc binds directly to a KIR promoter 1 Kb upstream of the conventional proximal promoter and leads to distal promoter transcription. We hypothesize that several additional factors play a role in NK cell education, including Notch, stromal, homeostatic and inflammatory activation signals (independent of specific NK cell receptors), and lastly, interaction with inhibitory receptors. Our developmental model with human cord blood stem cells is ideal because it recapitulates NK cell development but favors NK cell receptor negative hyporesponsive NK cells. Three specific aims are proposed: Specific Aim 1: NK cell commitment and acquisition of global function as a developmental event. Specific Aim 2: NK cell education after NK cell commitment. Specific Aim 3: Mechanisms of KIR acquisition. The significance of these studies is of high translational interest given our own work suggesting a role for NK cells in hematopoietic cell transplantation and as therapy for cancer. Understanding mechanisms of KIR transcription may ultimately allow us to manipulate NK cells with specificity. PUBLIC HEALTH RELEVANCE: PROJECT Natural killer (NK) cells are a type of circulating white blood cell which can kill cancer cells and virally infected targets. As these innate immune cells develop from hematopoietic stem cells they develop the ability to kill targets and secrete cytokines. In order to induce self tolerance and avoid damage to healthy tissues, NK cells are educated by "self" MHC molecules which bind inhibitory NK receptors. The main goal of this R01 renewal is to understand the stages of NK cell development, including the acquisition of NK cell receptors, and the regulation of NK cell function. A thorough understanding of these mechanisms may allow us to manipulate NK cells for therapeutic purposes to treat cancer.